Squaric Acid Derivatives as Inhibitors of Histone Deacetylase

ABSTRACT

This invention comprises the novel compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2  and X have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

This invention concerns compounds having histone deacetylase (HDAC) inhibiting enzymatic activity. It further relates to processes for their preparation, to compositions comprising them, as well as their use, both in vitro and in vivo, to inhibit HDAC and as a medicine, for instance as a medicine to inhibit proliferative conditions, such as cancer and psoriasis.

Nuclear histones are known as integral and dynamic components of the machinery responsible for regulating gene transcription and other DNA-templated processes such as replication, repair, recombination, and chromosome segregation. They are the subject of post-translational modifications including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation.

Histone deacetylase(s), herein referred to as “HDACs”, are enzymes that catalyze the removal of the acetyl modification on lysine residues of proteins, including the core nucleosomal histones H2A, H2B, H3 and H4. Together with histone acetyltransferase(s), herein referred to as “HATs”, HDACs regulate the level of acetylation of the histones. The balance of acetylation of nucleosomal histones plays an important role in transcription of many genes. Hypoacetylation of histones is associated with condensed chromatin structure resulting in the repression of gene transcription, whereas acetylated histones are associated with a more open chromatin structure and activation of transcription.

Several structurally related HDACs have been described and fall into two classes. Class I HDACs consist of HDAC 1, 2, 3 and 8 whereas class II HDACs consist of HDAC 4, 5, 6, 7, 9 and 10. Members of a third class of HDACs are structurally unrelated to the class I and class II HDACs. Class I/II HDACs operate by zinc-dependent mechanisms, whereas class III HDACs are NAD-dependent.

In addition to histones, other proteins have also been the substrate for acetylation, in particular transcription factors such as p53, GATA-1 and E2F; nuclear receptors such as the glucocorticoid receptor, the thyroid receptors, the estrogen receptors; and cell-cycle regulating proteins such as pRb. Acetylation of proteins has been linked with protein stabilization, such as p53 stabilization, recruitment of cofactors and increased DNA binding. p53 is a tumour suppressor that can induce cell cycle arrest or apoptosis in response to a variety of stress signals, such as DNA damage. The main target for p53-induced cell cycle arrest seems to be the p21 gene. Next to its activation by p53, p21 has been identified by virtue of its association with cyclin/cyclin-dependent kinase complexes resulting in cell cycle arrest at both G1 and G2 phases, its up-regulation during senescence, and its interaction with the proliferating cell nuclear antigen.

The study of inhibitors of HDACs indicates that they play an important role in cell cycle arrest, cellular differentiation, apoptosis and reversal of transformed phenotypes.

The inhibitor Trichostatin A (TSA), for example, causes cell cycle arrest at both G1 and G2 phases, reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukemia cells and others. TSA (and suberoylanilide hydroxamic acid SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., Nature, 401: 188-193, 1999).

Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g. liver fibrosis and liver chirrhosis. (Geerts et al., European Patent Application EP 0 827 742, published 11 Mar. 1998).

The pharmacophore for HDAC inhibitors consists of a metal-binding domain, which interacts with the zinc-containing active site of HDACs, a linker domain, and a surface recognition domain or capping region, which interacts with residues on the rim of the active site.

Inhibitors of HDACs have also been reported to induce p21 gene expression. The transcriptional activation of the p21 gene by these inhibitors is promoted by chromatin remodelling, following acetylation of histones H3 and H4 in the p21 promotor region. This activation of p21 occurs in a p53-independent fashion and thus HDAC inhibitors are operative in cells with mutated p53 genes, a hallmark of numerous tumours.

In addition HDAC inhibitors can have indirect activities such as augmentation of the host immune response and inhibition of tumor angiogenesis and thus can suppress the growth of primary tumors and impede metastasis (Mai et al., Medicinal Research Reviews, 25: 261-309).

In view of the above, HDAC inhibitors can have great potential in the treatment of cell proliferative diseases or conditions, including tumours with mutated p53 genes.

Patent application EP1472216 published on Aug. 14, 2003 discloses bicyclic hydroxamates as inhibitors of histone deacetylase.

Patent applications EP1485099, EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370, EP1485378 published on 18 Sep. 2003, amongst others, disclose substituted piperazinylpyrimidinylhydroxamic acids as inhibitors of histone deacetylase furthermore EP1485365 discloses R306465.

Patent application EP1492534 published on 9 Oct. 2003, discloses carbamic acid compounds comprising a piperazine linkage, as HDAC inhibitors.

Patent application EP 1495002 published on 23 Oct. 2003, disclose substituted piperazinyl phenyl benzamide compounds, as histone deacetylase inhibitors.

Patent application EP1523470 published on 29 Jan. 2004, discloses derivatives containing an alkyl linker between the aryl group and the hydroxamate, as histone deacetylase inhibitors.

Patent application EP1525199 published on 12 Feb. 2004, discloses (hetero)arylalkenyl substituted bicyclic hydroxamates, as histone deacetylase inhibitors.

Patent application EP1581484 published on 29 Jul. 2004, discloses derivatives of N-hydroxy-benzamide derivatives with anti-inflammatory and antitumour activity.

Patent application EP1585735 published on 29 Jul. 2004, discloses substituted aryl hydroxamate derivatives as histone deacetylase inhibitors.

Patent application EP1583736 published on 12 Oct. 2005, discloses carbomic acid compounds comprising an ester or ketone linkage as histone deacetylase inhibitors.

Patent application WO04/072047 published on 26 Aug. 2004, discloses indoles, benzimidazoles and naphhimidazoles as histone deacetylase inhibitors.

Patent application EP1608628 published on 30 Sep. 2004, discloses hydroxamates linked to non-aromatic heterocyclic ring systems as histone deacetylase inhibitors.

Patent application EP1611088 published on 28 Oct. 2004, discloses hydroxamate derivatives as histone deacetylase inhibitors.

Patent application EP1673349 published on 31 Mar. 2005, discloses benzimidazoles as histone deacetylase inhibitors.

Patent applications WO05/030704 and EP1663953 published on 7 Apr. 2005, discloses benzamides as histone deacetylase inhibitors.

Patent application EP1685094 published on 6 May 2005, discloses acylurea connected and sulfonylurea connected hydroxamates as histone deacetylase inhibitors.

Patent application EP1682538 also published on 6 May 2005, discloses biaryl linked hydroxamates as histone deacetylase inhibitors.

Patent application WO05/086898 published on 22 Sep. 2005, discloses hydroxamates linked to pentaheterocyclic ring systems as histone deacetylase inhibitors.

The compounds of the present invention differ from the prior art in structure, in their pharmacological activity and/or pharmacological potency.

The problem to be solved is to provide histone deacetylase inhibitors with high enzymatic activity.

The novel compounds of the present invention solve the above-described problem. The compounds of the present invention show excellent histone deacetylase inhibiting enzymatic activity. They can have a desirable pharmacokinetic profile and good solubility.

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts and the stereo-chemically isomeric forms thereof, wherein each X is independently N or CH;

-   R¹ and R² are independently selected from hydrogen, C₁₋₆alkyl, mono-     or di(C₁₋₆alkyl)amino, C₁₋₆alkyloxyC₁₋₆alkyl, phenyl,     phenylC₁₋₆alkyl, phenyl(cyclopropyl)C₁₋₆alkyl,     heterocyclylC₁₋₆alkyl, phenyloxyC₁₋₆alkyl, tetrahydronaphtalenyl, or     phenylaminoC₁₋₆alkyl; -   each phenyl or heterocyclyl is optionally substituted with one, two     or three substituents each independently selected from halo,     polyhaloC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy, phenyl or phenylalkyl; -   heterocyclyl in the above is furanyl, thienyl, pyrrolyl, pyrrolinyl,     pyrrolidinyl, oxopyrrolidinyl, dioxolyl, oxazolyl, thiazolyl,     imidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl,     pyrazolidinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl,     thiadiazolyl, pyranyl, pyridinyl, piperidinyl, dioxanyl,     morpholinyl, dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl,     pyrazinyl, piperazinyl, triazinyl, trithianyl, indolizinyl, indolyl,     indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl,     benzthiazolyl, purinyl, quinolizinyl, quinolinyl, cinnolinyl,     phthlazinyl, quinazolinyl, quinoxalinyl or naphthyridinyl.

The term “histone deacetylase inhibitor” or “inhibitor of histone deacetylase” is used to identify a compound, which is capable of interacting with a histone deacetylase and inhibiting its activity, more particularly its enzymatic activity. Inhibiting histone deacetylase enzymatic activity means reducing the ability of a histone deacetylase to remove an acetyl group from a histone. Preferably, such inhibition is specific, i.e. the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone at a concentration that is lower than the concentration of the inhibitor that is required to produce some other, unrelated biological effect.

As used in the foregoing definitions and hereinafter, halo is generic to fluoro, chloro, bromo and iodo; C₁₋₆alkyl defines straight and branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as, e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl, 1-methylethyl, 2-methylpropyl, 2-methyl-butyl, 2-methylpentyl and the like; polyhaloC₁₋₆alkyl defines C₁₋₆alkyl containing three identical or different halo substituents for example trifluoromethyl.

Pharmaceutically acceptable addition salts encompass pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The pharmaceutically acceptable acid addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid addition salt forms, which the compounds of formula (I) are able to form. The compounds of formula (I) which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids; or organic acids such as, for example, acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-amino-salicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

The term “acid or base addition salts” also comprises the hydrates and the solvent addition forms, which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.

The term “stereochemically isomeric forms of compounds of formula (I)”, as used herein, defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures, which are not interchangeable, which the compounds of formula (I) may possess. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of formula (I) both in pure form or in admixture with each other are intended to be embraced within the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprise those compounds of formula (I) wherein one or several nitrogen atoms are oxidized to the so-called N-oxide, particularly those N-oxides wherein one or more of the piperidine-, piperazine or pyridazinyl-nitrogens are N-oxidized.

Some of the compounds of formula (I) may also exist in their tautomeric forms. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.

Whenever used hereinafter, the term “compounds of formula (I)” is meant to include also the pharmaceutically acceptable addition salts and all stereoisomeric forms.

As used herein, the terms “histone deacetylase” and “HDAC” are intended to refer to any one of a family of enzymes that remove acetyl groups from the ε-amino groups of lysine residues at the N-terminus of a histone. Unless otherwise indicated by context, the term “histone” is meant to refer to any histone protein, including H1, H2A, H2B, H3, H4, and H5, from any species. Human HDAC proteins or gene products, include, but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10 and HDAC-11. The histone deacetylase can also be derived from a protozoal or fungal source.

A first group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

-   a) each X is N; -   b) each phenyl or heterocyclyl is optionally substituted with one,     two or three substituents each independently selected from halo,     polyhaloC₁₋₆alkyl, C₁₋₆alkyloxy, phenyl or phenylalkyl; -   c) heterocyclyl is furanyl, pyrrolidinyl, oxopyrrolidinyl or     pyridinyl.

A second group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

-   a) each X is N; -   b) R¹ and R² are independently selected from hydrogen, C₁₋₆alkyl or     phenyl; -   c) each phenyl or heterocyclyl is optionally substituted with phenyl     or phenylalkyl; -   d) heterocyclyl is pyrrolidinyl.

A group of preferred compounds consists of those compounds of formula (I) wherein each X is N; each phenyl or heterocyclyl is optionally substituted with one, two or three substituents each independently selected from halo, polyhaloC₁₋₆alkyl, C₁₋₆alkyloxy, phenyl or phenylalkyl; and heterocyclyl is furanyl, pyrrolidinyl, oxopyrrolidinyl or pyridinyl.

A group of more preferred compounds consists of those compounds of formula (I) wherein each X is N; R¹ and R² are independently selected from hydrogen, C₁₋₆alkyl or phenyl; each phenyl or heterocyclyl is optionally substituted with phenyl or phenylalkyl; and heterocyclyl is pyrrolidinyl.

Most preferred compound is compound No. 1, compound No. 2, and compound No. 3.

The compounds of formula (I) and their pharmaceutically acceptable salts and N-oxides and stereochemically isomeric forms thereof may be prepared in conventional manner. The starting materials and some of the intermediates are known compounds and are commercially available or may be prepared according to conventional reaction procedures generally known in the art.

Some preparation methods will be described hereinafter in more detail. Other methods for obtaining final compounds of formula (I) are described in the examples.

a) Hydroxamic acids of formula (I) may be prepared by reacting an intermediate of formula (II) with an appropriate acid, such as for example, trifluoro acetic acid. Said reaction is performed in an appropriate solvent, such as, for example, methanol or dichloromethane.

b) Intermediates of formula (II) may be prepared by reacting an intermediate of formula (III) with an intermediate of formula (IV) in the presence of appropriate reagents such as N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine, monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). The reaction may be performed in the presence of a base such as triethylamine, in a suitable solvent, such as, a mixture of dichloromethane and tetrahydrofuran.

c) Alternatively, intermediates of formula (II) may be prepared by reacting an intermediate of formula (IX) with the intermediate of formula (VII) and an intermediate of formula (VIII) in the presence of a suitable solvent such as ethanol.

d) Intermediates of formula (III) may be prepared by reacting an intermediate of formula (V) with an appropriate acidic solution, e.g. hydrochloric acid, or basic solution, e.g. hydrogen bromide or sodium hydroxide, in a suitable solvent e.g. an alcohol, such as ethanol or propanol.

e) Intermediates of formula (V) can be prepared by reacting an intermediate of formula (VI) with the intermediate of formula (VII) and an intermediate of formula (VIII) in the presence of a suitable solvent such as ethanol.

The compounds of formula (I) and some of the intermediates may have at least one stereogenic centre in their structure. This stereogenic centre may be present in an R or an S configuration.

The compounds of formula (I) as prepared in the hereinabove described processes are generally racemic mixtures of enantiomers, which can be separated from one another following art-known resolution procedures. The racemic compounds of formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated there from by alkali. An alternative manner of separating the enantiomeric forms of the compounds of formula (I) involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably if a specific stereoisomer is desired, said compound would be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.

The compounds of formula (I), the pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof have valuable pharmacological properties in that they have a histone deacetylase (HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth of cells, including transformed cells, by administering an effective amount of a compound of the invention. Abnormal growth of cells refers to cell growth independent of normal regulatory mechanisms (e.g. loss of contact inhibition). This includes the inhibition of tumour growth both directly by causing growth arrest, terminal differentiation and/or apoptosis of cancer cells, and indirectly, by inhibiting neovascularization of tumours.

This invention also provides a method for inhibiting tumour growth by administering an effective amount of a compound of the present invention, to a subject, e.g. a mammal (and more particularly a human) in need of such treatment. In particular, this invention provides a method for inhibiting the growth of tumours by the administration of an effective amount of the compounds of the present invention. Examples of tumours which may be inhibited, but are not limited to, lung cancer (e.g. adenocarcinoma and including non-small cell lung cancer), pancreatic cancers (e.g. pancreatic carcinoma such as, for example exocrine pancreatic carcinoma), colon cancers (e.g. colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), prostate cancer including the advanced disease, hematopoietic tumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acute myelogenous leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MDS), tumours of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, gliomas, benign tumour of the skin (e.g. keratoacanthomas), breast carcinoma (e.g. advanced breast cancer), kidney carcinoma, ovary carcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention may be used for other therapeutic purposes, for example:

-   -   a) the sensitisation of tumours to radiotherapy by administering         the compound according to the invention before, during or after         irradiation of the tumour for treating cancer;     -   b) treating arthropathies and osteopathological conditions such         as rheumatoid arthritis, osteoarthritis, juvenile arthritis,         gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis         and systemic lupus erythematosus;     -   c) inhibiting smooth muscle cell proliferation including         vascular proliferative disorders, atherosclerosis and         restenosis;     -   d) treating inflammatory conditions and dermal conditions such         as ulcerative colitis, Crohn's disease, allergic rhinitis, graft         vs. host disease, conjunctivitis, asthma, ARDS, Behcets disease,         transplant rejection, uticaria, allergic dermatitis, alopecia         greata, scleroderma, exanthema, eczema, dermatomyositis, acne,         diabetes, systemic lupus erythematosis, Kawasaki's disease,         multiple sclerosis, emphysema, cystic fibrosis and chronic         bronchitis;     -   e) treating endometriosis, uterine fibroids, dysfunctional         uterine bleeding and endometrial hyperplasia;     -   f) treating ocular vascularisation including vasculopathy         affecting retinal and choroidal vessels;     -   g) treating a cardiac dysfunction;     -   h) inhibiting immunosuppressive conditions such as the treatment         of HIV infections;     -   i) treating renal dysfunction;     -   j) suppressing endocrine disorders;     -   k) inhibiting dysfunction of gluconeogenesis;     -   l) treating a neuropathology for example Parkinson's disease or         a neuropathology that results in a cognitive disorder, for         example, Alzheimer's disease or polyglutamine related neuronal         diseases;     -   m) treating psychiatric disorders for example schizophrenia,         bipolar disorder, depression, anxiety and psychosis;     -   n) inhibiting a neuromuscular pathology, for example,         amylotrophic lateral sclerosis;     -   o) treating spinal muscular atrophy;     -   p) treating other pathologic conditions amenable to treatment by         potentiating expression of a gene;     -   q) enhancing gene therapy;     -   r) inhibiting adipogenesis;     -   s) treating parasitosis such as malaria.

Hence, the present invention discloses the compounds of formula (I) for use as a medicine as well as the use of these compounds of formula (I) for the manufacture of a medicament for treating one or more of the above mentioned conditions.

The compounds of formula (I), the pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof can have valuable diagnostic properties in that they can be used for detecting or identifying a HDAC in a biological sample comprising detecting or measuring the formation of a complex between a labelled compound and a HDAC.

The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances, etc. Examples of the radioisotopes include ¹²⁵I, ¹³¹I, ³H and ¹⁴C. Enzymes are usually made detectable by conjugation of an appropriate substrate which, in turn catalyses a detectable reaction. Examples thereof include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. The luminous substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the subject compounds may be formulated into various pharmaceutical forms for administration purposes.

To prepare the pharmaceutical compositions of this invention, an effective amount of a particular compound, in base or acid addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets.

Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, to aid solubility for example, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause a significant deleterious effect to the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient, calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amount from the test results presented hereinafter. In general it is contemplated that a therapeutically effective amount would be from 0.005 mg/kg to 100 mg/kg body weight, and in particular from 0.005 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 0.5 to 500 mg, and in particular 10 mg to 500 mg of active ingredient per unit dosage form.

As another aspect of the present invention a combination of a HDAC-inhibitor with another anticancer agent is envisaged, especially for use as a medicine, more specifically in the treatment of cancer or related diseases.

For the treatment of the above conditions, the compounds of the invention may be advantageously employed in combination with one or more other medicinal agents, more particularly, with other anti-cancer agents. Examples of anti-cancer agents are:

-   -   platinum coordination compounds for example cisplatin,         carboplatin or oxalyplatin;     -   taxane compounds for example paclitaxel or docetaxel;     -   topoisomerase I inhibitors such as camptothecin compounds for         example irinotecan or topotecan;     -   topoisomerase II inhibitors such as anti-tumour podophyllotoxin         derivatives for example etoposide or teniposide;     -   anti-tumour vinca alkaloids for example vinblastine, vincristine         or vinorelbine;     -   anti-tumour nucleoside derivatives for example 5-fluorouracil,         gemcitabine or capecitabine;     -   alkylating agents such as nitrogen mustard or nitrosourea for         example cyclophosphamide, chlorambucil, carmustine or lomustine;     -   anti-tumour anthracycline derivatives for example daunorubicin,         doxorubicin, idarubicin or mitoxantrone;     -   HER2 antibodies for example trastuzumab;     -   estrogen receptor antagonists or selective estrogen receptor         modulators for example tamoxifen, toremifene, droloxifene,         faslodex or raloxifene;     -   aromatase inhibitors such as exemestane, anastrozole, letrazole         and vorozole;     -   differentiating agents such as retinoids, vitamin D and retinoic         acid metabolism blocking agents (RAMBA) for example accutane;     -   DNA methyl transferase inhibitors for example azacytidine and         decitabine;     -   kinase inhibitors for example flavoperidol, imatinib mesylate or         gefitinib;     -   farnesyltransferase inhibitors;     -   other HDAC inhibitors     -   inhibitors of the ubiquitin-proteasome pathway for example         Velcade; or     -   Yondelis.

The term “platinum coordination compound” is used herein to denote any tumour cell growth inhibiting platinum coordination compound which provides platinum in the form of an ion.

The term “taxane compounds” indicates a class of compounds having the taxane ring system and related to or derived from extracts from certain species of yew (Taxus) trees.

The term “topisomerase inhibitors” is used to indicate enzymes that are capable of altering DNA topology in eukaryotic cells. They are critical for important cellular functions and cell proliferation. There are two classes of topoisomerases in eukaryotic cells, namely type I and type II. Topoisomerase I is a monomeric enzyme of approximately 100,000 molecular weight. The enzyme binds to DNA and introduces a transient single-strand break, unwinds the double helix (or allows it to unwind) and subsequently reseals the break before dissociating from the DNA strand. Topisomerase II has a similar mechanism of action which involves the induction of DNA strand breaks or the formation of free radicals.

The term “camptothecin compounds” is used to indicate compounds that are related to or derived from the parent camptothecin compound which is a water-insoluble alkaloid derived from the Chinese tree Camptothecin acuminata and the Indian tree Nothapodytes foetida.

The term “podophyllotoxin compounds” is used to indicate compounds that are related to or derived from the parent podophyllotoxin, which is extracted from the mandrake plant.

The term “anti-tumour vinca alkaloids” is used to indicate compounds that are related to or derived from extracts of the periwinkle plant (Vinca rosea).

The term “alkylating agents” encompass a diverse group of chemicals that have the common feature that they have the capacity to contribute, under physiological conditions, alkyl groups to biologically vital macromolecules such as DNA. With most of the more important agents such as the nitrogen mustards and the nitrosoureas, the active alkylating moieties are generated in vivo after complex degradative reactions, some of which are enzymatic. The most important pharmacological actions of the alkylating agents are those that disturb the fundamental mechanisms concerned with cell proliferation in particular DNA synthesis and cell division. The capacity of alkylating agents to interfere with DNA function and integrity in rapidly proliferating tissues provides the basis for their therapeutic applications and for many of their toxic properties.

The term “anti-tumour anthracycline derivatives” comprise antibiotics obtained from the fungus Strep. peuticus var. caesius and their derivatives, characterised by having a tetracycline ring structure with an unusual sugar, daunosamine, attached by a glycosidic linkage.

Amplification of the human epidermal growth factor receptor 2 protein (HER 2) in primary breast carcinomas has been shown to correlate with a poor clinical prognosis for certain patients. Trastuzumab is a highly purified recombinant DNA-derived humanized monoclonal IgG1 kappa antibody that binds with high affinity and specificity to the extracellular domain of the HER2 receptor.

Many breast cancers have estrogen receptors and growth of these tumours can be stimulated by estrogen. The terms “estrogen receptor antagonists” and “selective estrogen receptor modulators” are used to indicate competitive inhibitors of estradiol binding to the estrogen receptor (ER). Selective estrogen receptor modulators, when bound to the ER, induces a change in the three-dimensional shape of the receptor, modulating its binding to the estrogen responsive element (ERE) on DNA.

In postmenopausal women, the principal source of circulating estrogen is from conversion of adrenal and ovarian androgens (androstenedione and testosterone) to estrogens (estrone and estradiol) by the aromatase enzyme in peripheral tissues. Estrogen deprivation through aromatase inhibition or inactivation is an effective and selective treatment for some postmenopausal patients with hormone-dependent breast cancer.

The term “antiestrogen agent” is used herein to include not only estrogen receptor antagonists and selective estrogen receptor modulators but also aromatase inhibitors as discussed above.

The term “differentiating agents” encompass compounds that can, in various ways, inhibit cell proliferation and induce differentiation. Vitamin D and retinoids are known to play a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types. Retinoic acid metabolism blocking agents (RAMBA's) increase the levels of endogenous retinoic acids by inhibiting the cytochrome P450-mediated catabolism of retinoic acids.

DNA methylation changes are among the most common abnormalities in human neoplasia. Hypermethylation within the promotors of selected genes is usually associated with inactivation of the involved genes. The term “DNA methyl transferase inhibitors” is used to indicate compounds that act through pharmacological inhibition of DNA methyl transferase and reactivation of tumour suppressor gene expression.

The term “kinase inhibitors” comprises potent inhibitors of kinases that are involved in cell cycle progression and programmed cell death (apoptosis)

The term “farnesyltransferase inhibitors” is used to indicate compounds that were designed to prevent farnesylation of Ras and other intracellular proteins. They have been shown to have effect on malignant cell proliferation and survival.

The term “other HDAC inhibitors” comprises but is not limited to:

-   -   carboxylates for example butyrate, cinnamic acid,         4-phenylbutyrate or valproic acid;     -   hydroxamic acids for example suberoylanilide hydroxamic acid         (SAHA), piperazine containing SAHA analogues, biaryl hydroxamate         A-161906 and its carbozolylether-, tetrahydropyridine- and         tetralone-analogues, bicyclic aryl-N-hydroxycarboxamides,         pyroxamide, CG-1521, PXD-101, sulfonamide hydroxamic acid,         LAQ-824, LBH-589, trichostatin A (TSA), oxamflatin, scriptaid,         scriptaid related tricyclic molecules, m-carboxy cinnamic acid         bishydroxamic acid (CBHA), CBHA-like hydroxamic acids,         trapoxin-hydroxamic acid analogue, CRA-024781, R306465 and         related benzoyl- and heteroaryl-hydroxamic acids, aminosuberates         and malonyldiamides;     -   cyclic tetrapeptides for example trapoxin, apidicin,         depsipeptide, spiruchostatin-related compounds, RedFK-228,         sulfhydryl-containing cyclic tetrapeptides (SCOPs), hydroxamic         acid containing cyclic tetrapeptides (CHAPs), TAN-174s and         azumamides;     -   benzamides for example MS-275 or CI-994, or     -   depudecin.

The term “inhibitors of the ubiquitin-proteasome pathway” is used to identify compounds that inhibit the targeted destruction of cellular proteins in the proteasome, including cell cycle regulatory proteins.

For the treatment of cancer the compounds according to the present invention may be administered to a patient as described above, in conjunction with irradiation. Irradiation means ionising radiation and in particular gamma radiation, especially that emitted by linear accelerators or by radionuclides that are in common use today. The irradiation of the tumour by radionuclides can be external or internal.

The present invention also relates to a combination according to the invention of an anti-cancer agent and a HDAC inhibitor according to the invention.

The present invention also relates to a combination according to the invention for use in medical therapy for example for inhibiting the growth of tumour cells.

The present invention also relates to a combination according to the invention for inhibiting the growth of tumour cells.

The present invention also relates to a method of inhibiting the growth of tumour cells in a human subject which comprises administering to the subject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormal growth of cells, including transformed cells, by administering an effective amount of a combination according to the invention.

The other medicinal agent and HDAC inhibitor may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular other medicinal agent and HDAC inhibitor being administered, their route of administration, the particular tumour being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.

The platinum coordination compound is advantageously administered in a dosage of 1 to 500 mg per square meter (mg/m²) of body surface area, for example 50 to 400 mg/m², particularly for cisplatin in a dosage of about 75 mg/m² and for carboplatin in about 300 mg/m² per course of treatment.

The taxane compound is advantageously administered in a dosage of 50 to 400 mg per square meter (mg/m²) of body surface area, for example 75 to 250 mg/m², particularly for paclitaxel in a dosage of about 175 to 250 mg/m² and for docetaxel in about 75 to 150 mg/m² per course of treatment.

The camptothecin compound is advantageously administered in a dosage of 0.1 to 400 mg per square meter (mg/m²) of body surface area, for example 1 to 300 mg/m², particularly for irinotecan in a dosage of about 100 to 350 mg/m² and for topotecan in about 1 to 2 mg/m² per course of treatment.

The anti-tumour podophyllotoxin derivative is advantageously administered in a dosage of 30 to 300 mg per square meter (mg/m²) of body surface area, for example 50 to 25 mg/m², particularly for etoposide in a dosage of about 35 to 100 mg/m² and for teniposide in about 50 to 250 mg/m² per course of treatment.

The anti-tumour vinca alkaloid is advantageously administered in a dosage of 2 to 30 mg per square meter (mg/m²) of body surface area, particularly for vinblastine in a dosage of about 3 to 12 mg/m², for vincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine in dosage of about 10 to 30 mg/m per course of treatment.

The anti-tumour nucleoside derivative is advantageously administered in a dosage of 200 to 2500 mg per square meter (mg/m²) of body surface area, for example 700 to 1500 mg/m², particularly for 5-FU in a dosage of 200 to 500 mg/m², for gemcitabine in a dosage of about 800 to 1200 mg/m² and for capecitabine in about 1000 to 2500 mg/m² per course of treatment.

The alkylating agents such as nitrogen mustard or nitrosourea is advantageously administered in a dosage of 100 to 500 mg per square meter (mg/m²) of body surface area, for example 120 to 200 mg/m², particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m², for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carmustine in a dosage of about 150 to 200 mg/m², and for lomustine in a dosage of about 100 to 150 mg/m² per course of treatment.

The anti-tumour anthracycline derivative is advantageously administered in a dosage of 10 to 75 mg per square meter (mg/m²) of body surface area, for example 15 to 60 mg/m², particularly for doxorubicin in a dosage of about 40 to 75 mg/m², for daunorubicin in a dosage of about 25 to 45 mg/m², and for idarubicin in a dosage of about 10 to 15 mg/m² per course of treatment.

Trastuzumab is advantageously administered in a dosage of 1 to 5 mg per square meter (mg/m²) of body surface area, particularly 2 to 4 mg/m² per course of treatment.

The antiestrogen agent is advantageously administered in a dosage of about 1 to 100 mg daily depending on the particular agent and the condition being treated. Tamoxifen is advantageously administered orally in a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally in a dosage of about 60 mg once a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Anastrozole is advantageously administered orally in a dosage of about 1 mg once a day. Droloxifene is advantageously administered orally in a dosage of about 20-100 mg once a day. Raloxifene is advantageously administered orally in a dosage of about 60 mg once a day. Exemestane is advantageously administered orally in a dosage of about 25 mg once a day.

These dosages may be administered for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days.

In view of their useful pharmacological properties, the components of the combinations according to the invention, i.e. the other medicinal agent and the HDAC inhibitor may be formulated into various pharmaceutical forms for administration purposes. The components may be formulated separately in individual pharmaceutical compositions or in a unitary pharmaceutical composition containing both components.

The present invention therefore also relates to a pharmaceutical composition comprising the other medicinal agent and the HDAC inhibitor together with one or more pharmaceutical carriers.

The present invention also relates to a combination according to the invention in the form of a pharmaceutical composition comprising an anti-cancer agent and a HDAC inhibitor according to the invention together with one or more pharmaceutical carriers.

The present invention further relates to the use of a combination according to the invention in the manufacture of a pharmaceutical composition for inhibiting the growth of tumour cells.

The present invention further relates to a product containing as first active ingredient a HDAC inhibitor according to the invention and as second active ingredient an anticancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from cancer.

EXPERIMENTAL PART

The following examples are provided for purposes of illustration. Hereinafter, “EDC” is defined as N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine, monohydrochloride, “DCM” is defined as dichloromethane, “DMF” is defined as N,N-dimethylformamide, “EtOH” is defined as ethanol, “HOBT” is defined as 1-hydroxy-1H-benzotriazole, “MeOH” is defined as methanol, “TFA” is defined as trifluoroacetic acid and “THF” is defined as tetrahydrofuran.

A. Preparation of the Intermediate Compounds Example A1 a) Preparation of Intermediate 1

A mixture of 3,4-diethoxy-3-cyclobutene-1,2-dione (0.00035 mol) and [1,1′-biphenyl]-3-amine (0.00035 mol) in EtOH (5 ml) was stirred for 3 days at 80° C. and then 2-(1-piperazinyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0030 mol) was added. The reaction mixture was stirred for 2 days at 80° C. and was then cooled. The resulting precipitate was filtered off, washed with a small amount of EtOH and dried, yielding 0.085 g (50%) of intermediate 1.

b) Preparation of Intermediate 2

A mixture of intermediate 1 (0.000096 mol) in sodiumhydroxide 1N (4 ml), THF (15 ml) and MeOH (5 ml) was stirred for 20 hours at room temperature and then the reaction mixture was neutralised with 1N HCl (4 ml). The mixture was diluted with DCM (5 ml) and filtered through Extrelut® and then the filtrate was evaporated, yielding 0.033 g (75%) of intermediate 2.

c) Preparation of Intermediate 3

A mixture of intermediate 2 (0.000072 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.00026 mol), EDC (0.00023 mol) and HOBT (0.00022 mol) in triethylamine (0.2 ml) and THF/DCM (10 ml) was stirred for 2 days at room temperature, then the reaction mixture was diluted with water (2 ml) and filtered through Extrelut®. The filtrate was evaporated and the resulting residue was purified by reversed phase high-performance liquid chromatography (Hypercil C18) (standard gradient). The product fractions were collected and the solvent was evaporated, yielding 0.011 g of intermediate 3.

Example A2 a) Preparation of Intermediate 4

A mixture of 2-(1-piperazinyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.059 mol) in THF (0 ml) and sodium hydroxideIN (300 ml) to stand overnight at room temperature and then stirred. Hydrochloric acid 1N (300 ml) was added and the mixture was stirred for 10 minutes. Sodium carbonate (0.178 mol) was added and the resulting mixture was stirred for 10 minutes at room temperature, then 1-[[(9H-fluoren-9-ylmethoxy)carbonyl]oxy]-2,5-pyrrolidinedione (0.059 mol) was added in small portions and the reaction mixture was stirred at room temperature for 15 hours. The mixture was acidified with concentrated hydrochloric acid and the resulting precipitate was filtered off and dried (vac.), yielding 22.5 g (94%) of intermediate 4, melting point 218.5-221.2° C.

b) Preparation of Intermediate 5

Triethylamine (0.069 mol), then EDC (0.0303 mol) and HOBT (0.0303 mol) followed by O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0303 mol) were added to a mixture of intermediate 4 (0.0233 mol) in DCM/THF (500 ml) and the reaction mixture was stirred at room temperature for 18 hours. The mixture was diluted with DCM and washed with water. The organic layer was separated and washed with a 10% sodium carbonate solution. The separated organic layer was dried (MgSO₄), filtered off and the solvent was evaporated. The residue was purified by chromatography on silica gel (eluent: DCM/MeOH 100/0 to 97.5/2.5). The product fractions were collected and the solvent was evaporated, yielding 8.4 g (68%) of intermediate 5.

c) Preparation of Intermediate 6

A mixture of intermediate 5 (0.016 mol) in piperidine (0.040 mol) and DCM (200 ml) was stirred overnight at room temperature and the reaction mixture was extracted with water, then the aqueous layer was concentrated and co-evaporated with acetonitrile. The residue (3.5 g) was purified by chromatography on silica gel (eluent: DCM/(MeOH/NH₃) 95/5). The product fractions were collected and the solvent was evaporated, yielding 2.5 g (50%) of intermediate 6, melting point 70.8-93.9° C.

d) Preparation of Intermediate 7

A mixture of intermediate 6 (0.0032 mol), 3,4-diethoxy-3-cyclobutene-1,2-dione (0.00294 mol) and triethylamine (0.0032 mol) in EtOH (20 ml) was stirred for 30 min. at room temperature and then the reaction mixture was diluted with DCM (50 ml). The organic layer was separated, washed with water, dried (MgSO₄), filtered off and the solvent was evaporated. The residue was suspended in acetonitrile, then the resulting precipitate was filtered off, washed with a small amount of acetonitrile and dried (vac.), yielding 0.516 g (40.9%) of intermediate 7.

e) Preparation of Intermediate 8

A mixture of intermediate 7 (0.000058 mol), 1-(phenylmethyl)-3-pyrrolidinemethanamine (0.000115 mol) and triethylamine (0.00035 mol) in EtOH (10 ml) was stirred and refluxed for 3 days and then the solvent was evaporated. The residue was dissolved in DCM/DMF (4/1) (10 ml) and then Tris-(2-aminomethyl)-amine polystyrene HL (200-400 mesh), 1% DVB (Novabiochem 01-64-0170) (0.100 g) and methylisocyanate polystyrene HL(200-400 mesh), 2% DVB (Novabiochem 01-64-0169) (0.100 g) were added. The resulting mixture was shaken for 20 hours at room temperature and the resins were filtered off. The filtrate was evaporated and the obtained residue was used as such in the next reaction step, yielding 0.050 g of intermediate 8.

B. Preparation of the Final Compounds Example B1 Preparation of Compound 1

A mixture of intermediate 3 (0.0000198 mol) in TFA (0.2 ml) and DCM/MeOH (4 ml) was stirred for 2 days at room temperature and then the desired product was blown dry under N₂ atmosphere, yielding 0.010 g (100%) of compound 1.

Example B2 Preparation of Compound 2

A mixture of intermediate 8 (0.000018 mol) in TFA (0.5 ml) and DCM/MeOH (50/50) (10 ml) was stirred for 3 days at room temperature and then the reaction mixture was blown dry under N₂, yielding 0.010 g of compound 2.

Table F-1 lists the compounds that were prepared according to one of the above Examples.

TABLE F-1 (final compounds)

C. Pharmacological Example

The in vitro assay for inhibition of histone deacetylase (see example C.1) measures the inhibition of HDAC enzymatic activity obtained with the compounds of formula (I).

The solubility of a compound measures the ability of a compound to stay in solution. DMSO-stock solutions are diluted with a single aqueous buffer solvent in 3 consecutive steps. For every dilution turbidity is measured with a nephelometer. (see example C.2).

Example C.1 In Vitro Assay for Inhibition of Histone Deacetylase

The HDAC Fluorescent Activity Assay/Drug Discovery Kit of Biomol (cat.No: AK-500-0001) was used. The HDAC Fluorescent Activity Assay is based on the Fluor de Lys (Fluorogenic Histone deAcetylase Lysyl) substrate and developer combination. The Fluor de Lys substrate, comprises an acetylated lysine side chain. Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment with the Fluor de Lys developer produces a fluorophore.

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with 75 μM of substrate. The Fluor de Lys substrate was added in a buffer containing 25 mM Tris, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl₂.6H₂O at pH 7.4. After 30 min, 1 volume of the developer was added. The fluorophore was excited with 355 nm light and the emitted light (450 nm) was be detected on a fluorometric plate reader.

For each experiment, controls (containing HeLa nuclear extract and buffer), a blank incubation (containing buffer but no HeLa nuclear extract) and samples (containing compound dissolved in DMSO and further diluted in buffer and HeLa nuclear extract) were run in parallel. In first instance, compounds were tested at a concentration of 10⁻⁵M. When the compounds showed activity at 10⁻⁵M, a concentration-response curve was made wherein the compounds were tested at concentrations between 10⁻⁵M and 10⁻⁹M. All sample were tested 4 times. In each test the blank value was substracted from both the control and the sample values. The control sample represented 100% of substrate deactylation. For each sample the fluorescence was expressed as a percentage of the mean value of the controls. When appropriate IC₅₀-values (concentration of the drug, needed to reduce the amount of metabolites to 50% of the control) were computed using probit analysis for graded data. Herein the effects of test compounds are expressed as pIC₅₀ (the negative log value of the IC₅₀-value) (see Table F-2).

Example C.2 Kinetic Solubility in Aqueous Media

In the first dilution step, 10 μl of a concentrated stock-solution of the active compound, solubilized in DMSO (5 mM), was added to 100 μl phosphate citrate buffer pH 7.4 and mixed. In the second dilution step, an aliquot (20 μl) of the first dilution step was further dispensed in 100 μl phosphate citrate buffer pH 7.4 and mixed. Finally, in the third dilution step, a sample (20 μl) of the second dilution step was further diluted in 100 μl phosphate citrate buffer pH 7.4 and mixed. All dilutions were performed in 96-well plates. Immediately after the last dilution step the turbidity of the three consecutive dilution steps were measured with a nephelometer. Dilution was done in triplicate for each compound to exclude occasional errors. Based on the turbidity measurements a ranking is performed into 3 classes. Compounds with high solubility obtained a score of 3 and for this compounds the first dilution is clear. Compounds with medium solubility obtained a score of 2. For these compounds the first dilution is unclear and the second dilution is clear. Compounds with low solubility obtained a score of 1 and for these compounds both the first and the second dilution are unclear (see Table F-2).

TABLE F-2 lists the results of the compounds that were tested according to example C.1 and C.2. Enzymatic activity Solubility Compound pIC50 C.2. number C.1. score 1 7.7 2 6.9 3 7.6 4 7.2 5 7.4 6 7.2 7 7.2 3 8 7.2 9 7.0 10 7.1 11 7.0 12 7.2 3 13 7.4 14 6.9 15 6.8 16 7.3

D. Composition Example Film-Coated Tablets Preparation of Tablet Core

A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 g starch is mixed well and thereafter humidified with a solution of 5 g sodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 ml of water. The wet powder mixture is sieved, dried and sieved again. Then there is added 100 g microcrystalline cellulose and 15 g hydrogenated vegetable oil. The whole is mixed well and compressed into tablets, giving 10.000 tablets, each comprising 10 mg of a compound of formula (I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanol there is added a solution of 5 g of ethyl cellulose in 150 ml of dichloromethane. Then there are added 75 ml of dichloromethane and 2.5 ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten and dissolved in 75 ml of dichloromethane. The latter solution is added to the former and then there are added 2.5 g of magnesium octadecanoate, 5 g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspension and the whole is homogenated. The tablet cores are coated with the thus obtained mixture in a coating apparatus. 

1. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts and the stereo-chemically isomeric forms thereof, wherein each X is independently N or CH; R¹ and R² are independently selected from hydrogen, C₁₋₆alkyl, mono- or di(C₁₋₆alkyl)amino, C₁₋₆alkyloxyC₁₋₆alkyl, phenyl, phenylC₁₋₆alkyl, phenyl(cyclopropyl)C₁₋₆alkyl, heterocyclylC₁₋₆alkyl, phenyloxyC₁₋₆alkyl, tetrahydronaphtalenyl, or phenylaminoC₁₋₆alkyl; each phenyl or heterocyclyl is optionally substituted with one, two or three substituents each independently selected from halo, polyhaloC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy, phenyl or phenylalkyl; heterocyclyl in the above is furanyl, thienyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, oxopyrrolidinyl, dioxolyl, oxazolyl, thiazolyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyranyl, pyridinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, trithianyl, indolizinyl, indolyl, indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolinyl, cinnolinyl, phthlazinyl, quinazolinyl, quinoxalinyl or naphthyridinyl.
 2. A compound according to claim 1 wherein each X is N; each phenyl or heterocyclyl is optionally substituted with one, two or three substituents each independently selected from halo, polyhaloC₁₋₆alkyl, C₁₋₆alkyloxy, phenyl or phenylalkyl; and heterocyclyl is furanyl, pyrrolidinyl, oxopyrrolidinyl or pyridinyl.
 3. A compound according to claim 1 wherein each X is N; R¹ and R² are independently selected from hydrogen, C₁₋₆alkyl or phenyl; each phenyl or heterocyclyl is optionally substituted with phenyl or phenylalkyl; and heterocyclyl is pyrolidinyl.
 4. A compound selected from the group consisting of:


5. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim
 1. 6. A process of preparing a pharmaceutical composition as claimed in claim 5 wherein the pharmaceutically acceptable carriers and a compound as claimed in claim 1 are intimately mixed. 7-8. (canceled)
 9. A combination of an anti-cancer agent and a HDAC inhibitor as claimed in claim
 1. 10. A process for preparing a compound as claimed in claim 1, said process comprising reacting an intermediate of formula (II) with an appropriate acid yielding a hydroxamic acid of formula (I),


11. A method of treating in a subject a disorder related to proliferative diseases comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
 12. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim
 2. 13. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim
 3. 14. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient a therapeutically effective amount of a compound as claimed in claim
 4. 